1. Field of the Invention:
This invention relates to a separation and purification method for obtaining bovine lactoferrin from cow's milk in a highly pure state and in high yield.
Lactoferrin is an iron-binding protein found in externally secreted fluids such as milk. It is not only highly beneficial in the feeding of infants from a nutritional point of view, but also has the physiological effect of exerting a bacteriostatic action on pathogenic bacteria occurring in the intestines and exhibiting a high degree of iron requirement because of its characteristic iron-binding ability. In other word, lactoferrin may be said to be an important milkprotein which not only serves as a nutrient but also has a pharmacological significance as an antiinfective agent, similar to immunoglobulins and lysozyme present in milk.
2. Description of the Prior Art:
Conventionally, in view of the above characteristic properties of lactoferrin, a number of methods for separating lactoferrin from milk and purifying it have been proposed. However, since lactoferrin is a protein having a highly reactive molecular structure and is also apt to interact with other milkproteins, it has been difficult to isolate a highly pure form of lactoferrin easily and effectively according to the conventional methods.
For example, one conventional method for separating and purifying lactoferrin comprises providing skim milk from which fat has been separated, removing casein therefrom by isoelectric precipitation at pH 4.6, salting out the resulting whey fraction with ammonium sulfate, dialyzing the resulting fraction against deionized water, and then passing the retentate several times through an ion exchange resin (Gordon, Ziegler, Bash et al.: Biochim. Biophys. Acta, 60, 410-411, 1962; Merton L. Glove et al.: Biochim. Biophys. Acta, 100, 154-162, 1965; Johansson, B. G. et al.: Acta Chem. Scand., 23, 683, 1969). In addition, modifications of the above method include a method in which silica particles are used in place of the ion exchange resin (Japanese patent Laid-Open No. 28233/'83) and a method in which, after the retentate is passed through an ion exchange resin, the resulting product is further subject to copper affinity chromatography (Norihiro Kawakata, Yoshio Yoshino et al., Abstracts of Lectures at the 1983 Annual Meeting of the Japan Biochemical Society, p. 1053). However, these methods are all lacking in practical utility because they require complicated operations and an unduly long treating time
Moreover, since lactoferrin has the property of interacting with other proteins present in milk, the above methods using an ion exchange resin cannot avoid contamination of the product with immunoglobulins and other proteins present in milk. Accordingly, it is difficult in practice to obtain a highly pure form of lactoferrin. In addition, these methods are also disadvantageous in that the repeated treatment by salting-out and ion exchange not only causes a marked reduction in the recovery of lactoferrin but also makes it practically impossible to recover and reuse milkproteins other than lactoferrin and other milk components present in the residues obtained during the course of the separation and purification of lactoferrin.